(a) Field of the Invention
The invention relates in general to biologically important intracellular agonists and antagonists such as peptides and other proteins. More specifically, the present invention relates to modifying hydrophilic peptides corresponding to intracellular regions of receptors and effector proteins to form membrane-permeable hydrophobic derivatives enabling them to cross cell barriers without disrupting the cell membrane to interact with their intracellular targets.
(b) Description of Prior Art
Intracellular agonists are, by definition, molecules that, when introduced into cells, will take the place of endogenous ones and specifically activate distinct intracellular signalling pathways such as G-protein activation, adenylyl cyclases, and phospholipases. Intracellular antagonists are, on the other hand, molecules that oppose the effects of cell behaviour to certain stimuli. Most of the published studies have concentrated on the use of extracellular antagonists to inhibit or block induced cell activation both in vivo and in vitro.
Uncontrolled cell activation can be a cause of disease and inflammation and constitutes one of the manifestations of such diseases. Inflammation is a complex process that involves the action of a variety of factors such as chemokines and cytokines. Pro-inflammatory chemotactic factors and chemokines such as fMLP, C5a, IL-8, NAP-2, Go-, PAF, LTB4, and MIP-1 are essential in initiating the inflammatory cascade which may result in cell and tissue damage. Most of these factors and their receptors are well-characterized at the biological and molecular levels. Their interaction with inflammatory cells (neutrophils, monocytes/macrophages, lymphocytes) leads to the initiation of the activation of a complex array of intracellular biochemical events, several of which have been described in detail.
Earliest signalling events in chemokine activation is G-protein coupling to the intracellular loops of surface receptors activated by hormones and peptides. G-proteins (guanine nucleotide binding proteins) are integral part of regulating mechanisms that operate in all human cells. Impairing G proteins function can affect a cell's response to hormone or chemotactic peptides signals e.g. by interfering with intracellular metabolic pathways. G-proteins act as essential parts of transducing mechanisms by which hormones and neurotransmitters convey their signals through the plasma membrane of the cell and thus elicit appropriate intracellular responses leading to cell function. These signal transducing mechanisms comprise three distinct components:
1-a receptor protein with an extracellular binding site specific for a given agonist or hormone; PA1 2-a membrane-bound effector protein that when activated catalyzes the formation or facilitates the transport of an intracellular second messenger, such as adenyl cyclase which converts ATP to cyclic AMP (cAMP); and PA1 3-a protein which functions as a communicator between these two. PA1 a) converting temporary primary amines of a peptide to acetamides, thereby rendering the peptide so-converted less water-soluble; PA1 b) converting temporary carboxylic acid and alcohol groups of the peptide obtained from step a) to esters; and PA1 c) dissolving the peptide obtained from step b) in cyclodextrane or polyethylene glycol which are pharmaceutically acceptable for administration. PA1 AM: Acetoxymethyl esters; PA1 fMLP: formyl Met-Leu-Phe; PA1 IL-8: Interleukin 8; PA1 NAP-2: Neutrophil activating peptide 2; PA1 Gro-: Melanocyte growth-stimulatory activity; PA1 MIP-1: Macrophage inflammatory peptide 1-; PA1 PAF: Platelet activating factor; PA1 C5a: the small fragment of the 5th component of complement; PA1 DMF: Dimethylformamide; and PA1 LTB4: Leukotriene B4. PA1 ARDS: Adult respiratory disease syndrome.
G-proteins fulfil this function as communicator between receptor and effector proteins in the generation of intracellular responses to extracellular hormones and agonists. This knowledge facilitates the design of small peptides (&gt;5 amino acids) directed against regions of intracellular proteins which, once introduced into the cells, can specifically and selectively interact with their targets to alter (enhance or inhibit) cell activation. This can lead to the development of novel therapeutic strategies based on the use of peptides directed against intracellular regions of proteins.
In International Patent Application published under No. WO/94/23724 on Oct. 27, 1994 in the name of The Regents of the University of California, there is described acyloxyalkyl esters of phosphate-containing second messengers which are capable of permeating cell membranes. Once inside the cell, the esters derivatives undergo enzymatic conversion to the biologically active form of the second messenger. The main disadvantage of this method is its specificity towards a single signalling pathway.
The use of peptides as extracellular antagonists is widely used for many therapeutic drugs. There are three descriptions of such reagents discussed in U.S. Pat. Nos. 5,607,691 (Hale et al.), 5,350,681 (Iacobucci et al.), and 5,332,802 (Kelman et al.).
Hale et al. discusses the use of peptide modifiers and chemical linkers to improve the transport and delivery of pharmaceutical agents (including peptides to extracellular domains of receptors and do not include peptides directed towards intracellular parts of transmembrane and intracellular proteins.
Kelman et al. and Iacobucci et al. support Hale et al. by disclosing some chemical modification of amine and carboxyl groups on peptides directed against extracellular receptors. The list of peptides listed in these patents include only peptide ligands such as chemokines, peptidic agonist and/or antagonist against extracellular receptors. These patents however has never taught any modification for intracellular peptides that would make it obvious to any skilled in the art to carry out. Also, their suggested modification do not make the peptides claimed in their patent hydrophobic enough to cross the membrane efficiently. Another important point in the modification taught by Kelman et al. and Iacobucci et al. is that the modification of the peptide is not reversible and would be inactive against intracellular target proteins.
On the other hand, little success has been reported with the use of peptides as intracellular agonists or antagonists due to the hydrophilic nature of proteins and amino acids which renders them incapable of crossing cell membranes due to low interaction with the membrane lipids.
It would be highly desirable to be provided with a new method that enables the modification of low molecular weight molecules (peptides larger than 5 amino acids) from a hydrophilic non-permeable to a completely hydrophobic membrane-permeable form.